PROJECT SUMMARY Muscle injury is often accompanied by disruption of the muscle plasma membrane, and disruption of the muscle plasma membrane leads to muscle injury. The elongated nature of skeletal myofibers, while optimized for muscle contraction, places stress and strain on the sarcolemma through direct and lateral force transmission. Mutations in genes encoding dystrophin and its associated proteins are associated with a fragile plasma membrane that is more susceptible to rupture. Loss-of-function mutations in the dysferlin gene cause muscular dystrophy through abnormal muscle membrane trafficking, which includes defective sarcolemmal repair. An improved understanding of the membrane repair machinery of muscle will not only provide useful information for developing novel approaches to treat dysferlinopathies, but also will provide insight for muscle repair in many forms of muscle injury. Muscle injury occurs with disease, intense exercise, trauma, and surgery, and enhancing muscle repair may benefit each of these needs. Dysferlin, and the highly related myoferlin, are membrane-anchored proteins with multiple C2 domains, domains known to have protein and phospholipid binding properties. We used high-resolution microscopy and real time live imaging to define distinct subcomplexes within the sarcolemma repair machinery. The first subcomplex is the repair cap, which forms within seconds after membrane disruption. The repair cap is enriched for annexins, including annexins A6, A2, and A1. Immediately beneath this cap within the cytoplasm is an annexin-free zone enriched with filamentous actin. The repair cap is surrounded by a ring of shoulder proteins that include dysferlin, EHD proteins, BIN1 and others. The shoulder region is enriched for phosphatidylserine indicating the importance of phospholipid signaling for sarcolemmal repair. The repair cap and the shoulder ring are independent structures. We propose a model in which calcium and phospholipid signaling orchestrate the assembly of the repair complex in muscle by first organizing annexin caps. In this model, phospholipid signaling drives assembly of the ring of shoulder proteins, which requires their membrane-bending capacity. We will test this model of sarcolemmal repair using laser injury and high resolution imaging in isolated muscle fibers. We will also evaluate this model in the context of in vivo muscle disease and injury. The first aim will conduct experiments to delineate annexin assembly with focus on the role of calcium as a trigger for repair cap formation. The second aim concentrates on defining the role of dysferlin and its interaction with BIN1 and EHD proteins in forming the shoulder ring. The third aim outlines experiments to improve muscle membrane repair. These studies will decipher repair complex interactions with the goals of mitigating sarcolemmal injury and improving muscle repair.